Endothelium derived nitric oxide is an important regulator of cardiovascular homeostasis. In the past grant cycle, we have shown the importance of Akt1 in regulating the extent of peripheral and coronary atherosclerosis in a unique murine model leading to spontaneous myocardial infarction and sudden death. In vitro studies support the importance of Akt1 as a key survival pathway in all cell types relevant to atherogenesis including endothelium (EC), smooth muscle cells (VSM) and macrophages and in vivo bone marrow transfer experiments suggested the coronary atherosclerosis in the ApoE-/-/Akt1-/- mice was of vascular origin. In the second area, we have dissected the role of the Akt1-eNOS axis as an example of specific kinase-substrate relationship promoting arteriogenesis and angiogenesis, in vivo. We have generated conditional Akt1-/- knockout mice and propose to delete this kinase from EC or VSM lineages and to examine the importance of Akt1 in each cell type during the development of atherosclerosis as well as in models of post-natal angiogenesis. In addition, we show that eNOS is required for VEGF induced changes in EC barrier function and that eNOS derived NO is a critical downstream pathway integrating VEGF signaling to VE- cadherin (VE-cad) function. Mechanistically, we will test the idea that Akt1 dependent phosphorylation of eNOS leads to NO dependent changes in paracellular permeability and vascular leakage, a critical component of the angiogenic response. We hypothesize that the Akt1 dependent phosphorylation of eNOS governs the production of NO to regulate macrovascular (atheroprotection) and microvascular (angiogenesis and permeability) functions in the cardiovascular system. As a corollary to this hypothesis, we predict that modulating eNOS phosphorylation will delay atherosclerosis and preserve regional blood flow and angiogenesis. To test these hypotheses, we will: 1.dissect the role of Akt1 regulating macrovascular (atherogenesis) and microvascular (angiogenesis) function by conditional deletion of Akt1 in vascular cells; 2. decipher the importance of eNOS phosphorylation on S1176 using knockin mice expressing either constitutively active (S1176D eNOS) or less active eNOS (S1176A eNOS) and characterize the specific role of Akt1- dependent eNOS phosphorylation on S1176 in regulating atherogenesis; and 3. elucidate the mechanisms whereby eNOS-derived NO regulates endothelial cell barrier functions, specifically focusing on how NO modulates VE-cad mediated changes in junctional permeability. Collectively, this work will facilitate our understanding of the importance of eNOS phosphorylation in atherogenesis and angiogenesis and provide insights into the molecular machinery required for eNOS regulation using a multi-disciplinary approach and state-of-the-art genetic mouse models.